Amylostereum Laevigatum Associated with the Japanese Horntail, Urocerusjaponicus

421

Mycoscience 38: 421-427, 1997

Amylostereum laevigatum associated with the Japanese horntail, Urocerusjaponicus

Masanobu Tabata 1~ and Yasuhisa Abe z)

1~ Shikoku Research Center, Forestry and Forest Products Research Institute, 915, Asakura-tei, Kochi 780, Japan 2~ Forestry and Forest Products Research Institute, P. O. Box 16, Tsukuba, Ibaraki 305, Japan

Accepted for publication 30 October 1997

The fungus associated with the Japanese horntail, Urocerus japonicus, in Kochi, Kagawa and Ehime Prefectures was studied. Cultures isolated from the mycangia of 113 adult females of the horntail showed the same cultural characteristics. Four of basidiocarps found on felled logs of Cryptomeriajaponica were identified as Amylostereumlaevigatum based on morphological characteristics. This was the first record of A. laevigatum from Japan. The cultures isolated from the basidiocarps had the same cultural characteristics as those from the mycangia of U.japonicus. One mycangial isolate produced basidiocarps on artificially inoculated stem segments of Cr. japonica after a 6-too incubation and was identified as A. laevigatum. One isolate from the basidiocarps of A. laevigatum and one from the mycangium of U. japonicus were artificially inoculated into five trees each of Chamaecyparis obtusa and Cr. japonica. The wood of all inoculated trees showed discoloration, with no difference in shape and pattern of discoloration between the two isolates. The inoculated fungi were reisolated from the areas of discoloration in the inoculated trees.

Key Words- Amylostereum laevigatum; Japanese horntail; symbiont; Urocerusjaponicus; wood discoloration.

Of the five recorded species of Amylostereum (Chamuris, quired to identify the symbionts exactly by teleomorph, 1988), onlyA, areolatum (Fr.: Fr.) Boidin and A. chailletii as fungal species other than A. areolaturn and A. chailletii (Pers.: Fr.) Boidin are known to be Sirex and Urocerus may be associated with horntails. For these reasons, we symbionts (Gaut, 1969, 1970; Sano et al., 1995). studied the fungus in the mycangia of Japanese horntails When the female of Urocerus japonicus Smith that emerged from felled logs of Ch. obtusa and Cr. (Japanese horntail) oviposits in the wood of host trees, japonica in Kochi, Kagawa, and Ehime Prefectures, Chamaecyparis obtusa (Sieb. et Zucc.) Endlicher and Shikoku District. The objective of this paper is to deter- Cryptomeriajaponica (L. f.) D. Don, the fungus is also in- mine the species of the fungus isolated from the mycan- oculated into the wood of these trees together with eggs gia of U. japonicus by teleomorph and cultural charac- (Kanamitsu, 1978; Okuda, 1989; Sano, 1992). After in- teristics. Furthermore, we examined the pathogenicity oculation, wood discoloration occurs (Okuda, 1989; of isolates from horntail and basidiocarp by means of in- Sano, 1992). When the eggs are deposited into the oculation experiments. stem in several directions, stellate discoloration is formed in the cross section of the stem (Okuda, 1989; Sano, Materials and Methods 1992). Discoloration in wood of Ch. obtusa and Cr. japonica extends up to 14-76 cm in the longitudinal Isolation from horntails Approximately 1,000 logs (7- direction, centered on the oviposition site (Nishiguchi et 17 cm in diam, 1-2 m long) of Ch. obtusa and Cr. japoni- al., 1981; Okuda, 1985). Wood discoloration of Ch. ob- ca, which appeared to have been attacked in the previous tusa and Cr. japonica caused by the Japanese horntail year by the horntail, were collected from 21 plantations and Amylostereum sp. has become problematic at planta- in Kochi, Kagawa and Ehime Prefectures (Table 1). tions in Shikoku, Kinki, Chugoku and other districts of The logs were brought into outdoor cages at the Shikoku Japan (Okuda, 1989; Sano, 1992; Suto, 1994). Research Center of the Forestry and Forest Products Kanamitsu (1978) reported A. chailletii to be carried Research Institute during the months of April and May in in the mycangia of females of U. japonicus, which is 1993, 1994, 1995 and 1996. Newly emerged horn- widely distributed in Japan (Takeuchi, 1962). Sano et tails were caught, and only the females were put in the al. (1995) reported the identification of the fungus isolat- freezer at -20~ for 1 5-30 min to paralyze them before ed from the mycangia of horntails in Mie Prefecture by dissection under a stereoscopic microscope for removal cultural characteristics. However, there have been no of their mycangia. Fungi taken from the mycangia were reports about identification of the fungus based on mor- mounted in lactophenol and observed under a light micro- phology of basidiocarps (teleomorph) induced from the scope. cultures isolated from the mycangia. Studies are re- Mycangia were also excised individually from horn- 422 M. Tabata and Y. Abe

Table 1. Origin of the Japanese horntail, Urocerusjaponicus, used to isolate the fungi from the mycangia.

Number of emerged horntails Locality of collection Date of collection Tree species Number of isolates Male Female

Hirotad), Ehimea) April 1996 Cr. japonica 5 1 1 Tsushimac), Ehimea) May 1994 Cr. japonica 6 0 0 Ayautael, Kagawaa) May 1994 Ch. obtusa 0 1 1 Ookawac~, Kagawaa) May 1994 Ch. obtusa 6 2 2 Oonoharac), Kagawaa) May 1994 Ch. obtusa 3 0 0 Ikegawac), Kochia) April 1996 Cr. japonica 0 2 2 Kitagawad), Kochi a) April 1994 Cr. japonica 0 5 5 Motoyamac), Kochia) April 1995 Cr. japonica 0 19 19 Ookawad), Kochia) April 1996 Cr. japonica 3 1 1 Oonomid), KochP ) April 1995 Cr. japonica 6 0 0 Ootoyol c), Kochia) May 1994 Cr. japonica 27 50 50 Ootoyo2c), Kochial April 1995 Cr. japonica 0 4 4 Ootoyo3c), KochP ) April 1996 Ch. obtusa 0 1 1 Sakawal c), Kochia) May 1993 Cr. japonica 10 1 1 Sakawa2c), Kochial May 1996 Ch. obtusa 0 5 5 Susaki b), Kochi a) April 1994 Ch. obtusa 0 3 3 Taishooc), Kochial April 1996 Ch. obtusa 5 1 1 Tosalcl, Kochial April 1994 Cr. japonica 6 0 0 Tosa2c~, Kochia) May 1996 Cr. japonica 0 4 4 Tosayamadac), Kochia) May 1996 Cr. japonica 0 9 9 Umaji d), Kochia) April 1994 Cr. japonica 5 4 4 Total 82 113 113 a) Prefecture; b) city; c) town; d) village. tails with sterilized forceps and put on potato-dextrose Forestry and Forest Products Research Institute. agar (PDA) in Petri plates. The Petri plates were incubat- Cultural characteristics A total of 113 cultures (Table 1) ed at 20~ for 4-7 d and mycelia growing from mycangia isolated from mycangia of individual female horntails and were isolated. All the cultures used are stored at the 3 cultures (FD-4, 115, 127, Table 2) isolated from the Shikoku Research Center of the Forestry and Forest basidiospores of the three basidiocarps were grown on Products Research Institute. PDA in Petri plates at 25~ and their cultural charac- Examination of basidiocarps on the felled logs Basidio- teristics were recorded and assigned according to the key carps occurring on four felled logs were collected at four pattern of Stalpers (1978). Cr. japonica plantations in Kochi Prefecture, on 16 Nov. The effect of temperature on mycelial growth was and 3 Dec. 1994 at Ootoyo Town, on 11 May 1995 at studied for four isolates (FD-1-4, Table 2) using 9-cm Niyodo Village, and on 20 Nov. 1996 at Ookawa Village. Petri plates containing 20 ml of PDA. A 4-mm plug cut The basidiocarps were examined morphologically and cul- from the colony of the test fungus on PDA was placed tures were isolated from basidiospores. All the isolates centrally on each plate. Inoculated plates were incubat- used are stored at the Shikoku Research Center of the ed at a test temperature from 0 to 35~ at intervals of

Table 2. Origin of 6 Amylostereum laevigatum isolates used to study the mycelial growth response to temperature and cultural characteristics.

Isolate Locality of collection Date of collection Origin FD-1 Sakawa~), KochP ~ 5 Jul. 1993 Mycangium of U. japonicus FE)-2 Ootoyob), Kochial 11 Aug. 1994 Mycangium of U. japonicus FD-3 TosayamadahI, Kochia~ 3 Aug. 1994 Mycangium of U. japonicus FD-4 Ootoyob), Kochia) 10 Dec. 1994 Basidiospores of A. laevigatum FD-115 Ootoyob), Kochial 10 May 1996 Basidiospores of A. laevigatum FD-127 Ookawac), KochP ) 29 May 1996 Basidiospores of A. laevigatum a) Prefecture; b) town; c) village. Amylostereum laevigatum associated with horntail 423

5~ with ten replicates for each temperature. Colony growth, the same color of colony, the same hyphal sys- size was measured along two diam at right angles every tem, and hyphae with clamp connections. Colonies 2 d and radial growth of mycelium was calculated. were white to creamy-brown and produced cottony to Inoculation to stem segments The logs (3-5 cm in diam) pelliculate mycelia on PDA (Fig. 8a). from fresh Cr. japonica stems were cut into segments of Basidiocarps of the genus Amylostereum were found 11-12 cm in length, and four holes (ca. 2.5 mm in diam among the collected basidiocarps formed on the bark of and ca. 1 cm long) were drilled in each segment. Four four Cr. japonica logs. They were pale brown and strict- stem segments were put into plastic bags singly and au- ly resupinate. Basidiospores measured 7-10 x 3-4 #m. toclaved at 121~ for 1 h. Sterilized toothpicks (ca. The fungus was identified as A. laevigatum based on 2 mm in diam and ca. 1 cm long) were put on PDA plates morphological characteristics. Three cultures (basidio- inoculated with a mycangial isolate (FD-2) and incubated spore isolates) were isolated successfully from the at 25~ in darkness for 3 wk. Cultured toothpicks were basidiospores of the three collected basidiocarps of A. aseptically put into the holes of segments with sterilized laevigatum. Basidiospore isolates had almost the same forceps. Inoculation was performed on 20 February cultural characteristics as the mycangial isolates. The 1995. The plastic bags were sealed and placed at the morphological and cultural characteristics of the fungus corner of the laboratory to the production of fruit bodies. were as follows. Inoculation of trees Five to seven yr-old trees of Ch. ob- Description tusa (4.0-6.0 m high, 4-7 cm in diam at breast height) Amylostereum laevigatum (Fr.) Bold., Rev. Mycol. 23: and 5 yr-old trees of Cr. japonica (4.1-5.0m high, 345. 1958. Figs. 3, 9-11 4-6 cm in diam at breast height) were used. They were Basidiocarps strictly resupinate, attached tightly to planted at the Shikoku Research Center of the Forestry the substrate, forming patches 3 to 15 cm in extent, and Forest Products Research Institute. One isolate 75-300 ~m thick (Fig. 9), hymenial surface smooth, pale from the mycangium of a Japanese horntail (FD-1) and brown, sometimes cracked when dry. Hyphal system one from a basidiocarp (FD-4) were used for inoculation. monomitic, generative hyphae hyaline, thin to thick- Toothpicks cultured as described above were used as in- walled with clamps, 3-4/~m wide. Cystidia present in ocula. Three holes (ca. 2.5mm in diam and ca. 1 cm the surface and inside of the hymenium, pale brown, long) were drilled at the height of 1.2 m in the stem of thick-walled, clavate with acute or somewhat rounded each tree. Cultured toothpicks (FD-1,4) and a sterilized edge, encrusted with granular matter, 23-50 x 3-7 #m, toothpick (control) were put into the holes and covered 11-25 x 3-7 pm in encrusted part (Fig. 11 ). Basidia sub- with plastic film (Parafilm) and adhensive tape. Five clavate to subcylindrical (Fig. 10a), 35-40x5-5.5#m trees each of the two tree species were inoculated on 29 with 4-sterigmata. Basidiospores ellipsoid to cylindrical, July 1996. Inoculated trees were cut down and exam- (6.5-)7-10x 3-4 #m, hyaline, smooth, thin-walled, and ined 1 mo after inoculation. The maximum extent of dis- amyloid (Fig. lOb). coloration in the wood was measured in the longitudinal, Cultural characteristics: Colonies cottony, finally pel- tangential, and radial directions, and results were ex- liculate, white to yellowish-brown on PDA (Fig. 8b) and pressed as the range and average extent of discoloration with offensive odor. Reverse pale brown. Cystidia pale of 15 inoculation points in five trees. Three inoculated brown, thick-walled, clavate, with acute or somewhat trees each of Ch. obtusa and Cr. japonica were used for rounded edge, encrusted with granular matter, isolation of the infecting fungus. Three to nine pieces of 28-88x3-6pm. Hyphae hyaline with clamps, thin- wood (3x4x 3mm) were taken from the areas of dis- walled. Marginal hyphae 2-5.5~m wide. Aerial coloration, treated with 70% ethanol solution for 30 min hyphae 1-4 #m wide. Key patterns of the culture were: and sodium hypochlorite solution (about 1% available 1,2, 3, 6, 13, 21, (22), 24, 30, (31), (35), (36), 38, 39, chlorine) for 2 min, then washed with sterilized water 44, 45, 48, (51), 52, 53, (54), (58), (60), 72, (83), 90, (twice), placed on PDA in Petri plates and incubated at 94. 20~ in darkness for 3 wk. Specimen examined: SFM 1; Ootoyo; Kochi; on bark of felled log of Cr. japonica; 16 Nov. 1994. SFM2; Results Ootoyo; Kochi; on bark of felled log of Cr. japonica; 3 Dec. 1994. SFM3; Niyodo; Kochi; on bark of felled log The fungus associated with horntails and felled logs A of Cr. japonica; 11 May 1995. SFM4; Ookawa; Kochi; total of 82 adult males and 113 adult females of U. on bark of felled log of Cr. japonica; 20 Nov. 1996. The japon/cus emerged from the collected logs during the specimens are stored at the Shikoku Research Center of period from mid-July to early September in 1993, 1994, the Forestry and Forest Products Research Institute. 1995 and 1996 (Table 1 ). The mycangia (Fig. 1 ) of the Mycelial growth of mycangial isolates and a basidiospore adult females (Fig. 2) were filled with a mass of hyphal isolate Figure 12 shows the effect of temperature on fragments (Fig. 7). The hyphal fragments were hyaline mycelial growth of the fungi from the mycangia of in color, 12.5-280/~m long and 2.5-8/~m wide. They Japanese horntails (FD-1-3) and from the basidiospores consisted of one to six cells and had clamp connections of a collected basidiocarp of A. /aevigatum (FD-4). at the septum. Mycelia were isolated from all the adult Mycelial growth occurred at temperatures between 5~ females, yielding a total of 113 cultures (mycangial iso- and 30~ in the four isolates. Maximum mycelial IAtp__~l ITAhlA II All P_llltllrFe. h~lPI th~ ~m~ rnvn~li~l nrnu~lth nr, Pllrr~tq ~t 9C1oc ~ in I::r~_l ~n~ 9 ~n~ ~t 9ROP in 424 M. Tabata and Y. Abe

Fig. 1. Anatomy of abdomen of Urocerusjaponicus; arrows show the mycangia. Fig. 2. Adult female of U. japonicus. Scale bar= 1 cm. Fig. 3. Basidiocarp of Amylostereum laevigatum on Cryptomeriajaponica bark collected in the field (SFM3). Scale bar= 1 cm. Fig. 4. Basidiocarp produced on the stem segment of Cr, japonica artificially inoculated with mycangiel isolate (FD-2). Scale bar= 1 cm. Fig. 5. Wood discoloration in the cross section of the stem of Chamaecyparis obtuse inoculated with the fungus from the mycangia of U. japonicus (a) and the fungus from the basidiospores ofA./aevigatum (b), and a sterilized toothpick (c). Fig. 6. Wood discoloration in the cross section of the stem of Cr. japonica inoculated with the fungus from the mycangia of U. japonicus (a) and the fungus from the basidiospores of A. laevigatum (b), and a sterilized toothpick (c). Amylostereum laevigatum associated with horntail 425

Fig. 7. Hyphal fragments with clamp connections (arrows) stored in the mycangia of U, japonicus. Scale bar= 5/~m. Fig. 8. Culture derived from the mycangia of U. japonicus (FD-2, a); culture from A. laevigatum collected on a Cr. japonica log (FD- 4, b). Both cultures were grown on PDA at 25~ in darkness for 1 wk. Figs. 9-11. Amylostereum laevigatum collected on Cr. japonica log, SFM3. 9. Section of the basidiocarp. 10. Basidium (a) and basidiospores (b). 11. Cystidia (arrows). Scale bars: 9=50pm; 10, 1 1 =lO/~m.

FD-3 and 4. The pattern of growth was same for the tusa than Cr, japonica, spindle-form in the cross section, four isolates. and ovariform in the longitudinal section. Table 3 shows Inoculation to the stem segments Fruit bodies were the extent of discoloration in Ch. obtusa and Cr. japonica produced on stem segments 6 mo after the inoculation of one month after inoculation. In the longitudinal direc- isolate FD-2. They were strictly resupinate, attached tion, the average discoloration in Ch. obtusa was more tightly to the substrate and formed patches 1-10 cm in than twice that in Cr. japonica, and the maximal discolo- extent, 75-225 pm thick. Hymenial surface was ration in wood inoculated with the basidiospore isolate smooth and pale brown (Fig. 4). reached 28.5 cm and 1 5.7 cm, respectively. There was Basidiospores produced on stem segments meas- no difference in the pattern or extent of discoloration be- ured 6.5-9x 3-3.5/~m, and microscopic characteristics tween the basidiospore isolate and the mycangial isolate of basidiocarps were almost identical with those of the in trees of the same species. The inoculated fungi were specimens (SFM 1-4). reisolated from the areas of discoloration of all the inocu- Inoculation of trees Wood discoloration occurred in all lated trees but not from controls. Pestalotiopsis sp. was trees inoculated with isolates (Figs. 5, 6). Discoloration also isolated from the areas of discoloration of the inocu- was reddish brown to pale brown, being paler in Ch. oh- lated trees and all controls. 426 M. Tabata and Y. Abe

12 --~ FD-1 -~- FD-2 ~ _ \ \ i~ 10 + FD-3 /~/~..~"--~.~ \ \

~ 6

~ 4

0 0 5 l0 15 20 25 30 35

Temperature (~ Fig. 12. Effect of temperature on mycelial growth of three isolates from the mycangia of Urocerusjaponicus (FD-1-3) and one isolate from the basidiospores of Amylostereum laevigatum (FD-4).

Table 3. Extent of discoloration in wood of Chamaecyparis obtusa and Cryptomeriajaponica inoculated with Amylostereurn laevigaturn isolates.

Longitudinal direction (cm) Tangential direction (cm) Radial direction (cm) Inoculum Tree species Range of Average Range of Average Range of Average discoloration discoloration discoloration FD-1 Ch. obtusa 12.4-23.3 19.6 0.5-0.8 0.6 1.6-2.4 2.1 FD-4 Ch, obtusa 14.0-28.5 19.5 0.4-0.7 0.6 1.6-2.4 2.0 Control Ch. obtusa 0.7- 1.5 1.1 0.2-0.3 0.2 0.8-1.7 1.2 FD-1 Cr. japonica 6.2-10.3 7.6 0.4-0.6 0.4 1.4-2.2 1.8 FD-4 Cr. japonica 5.1-15.7 9.8 0.4-0.5 0.5 1.2-2.3 1.8 Control Cr. japonica 0.9- 2.8 1.4 0.2-0.3 0.3 1.1-1.8 1.3

Discussion cerus species (Gaut, 1969, 1970). Eriksson and Ryvarden (1973), Breitenbach and Kr~nzlin (1986), and All the mycangial isolates from 113 adult females of the Chamuris (1988) state that the basidiocarps of A. Japanese horntail produced the same colonies and had chailletii are resupinate to effuso-reflexed with a narrow the same microscopic characteristics, suggesting that a margin and that the hyphal system is dimitic. Reported single species of fungus is associated with the horntail in basidiospore sizes of A. chailletii are 6-7.5 x 2.5-3/~m Kochi, Kagawa and Ehime Prefectures in Shikoku Dis- (Eriksson and Ryvarden, 1973), 5.5-7x 2.5-3/~m trict. The cultural characteristics of mycangial isolates (Breitenbach and Kr~inzlin, 1986), and 5-8x2-3.5 coincided closely with those of basidiospore isolates. (-4) f~m (Chamuris, 1988). The size of basidiospores of The basidiocarps collected on the felled logs of Cr. japoni- basidiocarps collected on the felled logs and produced on ca in the field and those produced on the Cr. japonica the stem segments in this study partially overlaps with stem segments in the laboratory were both resupinate that of A. chailletii, but A. laevigatum has a monomitic and had monomitic hyphal system. The basidiocarps hyphal system and its basidiocarps never become reflex- collected on the felled logs were slightly larger, but their ed. From these facts, A. laevigaturn was concluded to morphological characteristics accorded closely with be the symbiotic fungus of the Japanese horntail in those of basidiocarps produced on the stem segments. Kochi, Kagawa and Ehime Prefectures of Shikoku Dis- There was little difference in the size of basidiospores be- trict. The fungus is distributed in Canada, France, Nor- tween the basidiocarps of mycangial isolates and those way, Sweden, Switzerland and USA (Boidin and Lanque- of a basidiospore isolate. Thus, the fungus isolated from tin, 1984; Breitenbach and Kr~inzlin, 1986; Eriksson and the mycangia of U. japonicus is conspecific with the one Ryvarden, 1973; Ginns and Lefebvre, 1993). It has producing basidiocarps on the felled logs of Cr. japonica. never been recorded as a symbiont of Sirex or Urocerus Amylostereum laevigatum is different from A. species but is known to occur on the hosts of Abies, chailletii, which is known as a symbiont of Sirex and Uro- Juniperus, Taxus, and Thuja (Boidin and Lanquetin, Amylostereum laevigaturn associated with horntail 427

1984; Breitenbach and Kr~inzlin,1986; Eriksson and Boidin, J. and Lanquetin, P. 1984. Le genre Amylostereum Ryvarden, 1973; Ginns and Lefebvre, 1993). In Japan, (Basidiomycetes) intercompatibilit~s partielles entre es- Kitajima (1938) and Hayashi (1974) have stated that the p~ces allopatriques. Bull. Soc. Mycol. France 100: fungus occurs on the bark of Ch. obtusa. The former 211-236. records only the scientific name of the fungus but gives Breitenbach, J. and Kr~inzlin, F. 1986. Fungi of Switzerland, vol. 2. Non-gilled fungi. Verlag Mykologia, Lecerne. no concrete description. Therefore, we were unable to Chamuris, G. 1988. The non-stipitate stereoid fungi in the confirm whether it is A, laevigatum or not. The latter northeastern United States and adjacent Canada. Mycolo- give a full description of the fungus. We examined the gia Mem. No. 14. Stuttgart. specimen (10335-F, TFM) in more detail and found that it Eriksson, K. and Ryvarden, L. 1973. The Corticiaceae of North was a different species. This is the first report on A. Europe, vol. 2. Fungiflora, Oslo. laevigatum associated with the horntail in the world and Francke-Grosmann, H. 1939. 0ber das Zusammenlebeb von the first record of A. laevigatum from Japan. Holzwespen (Siricinae) mit Pilzen. Z. Angew. Entmol. 25: Francke-Grosmann (1939), Stillwell (1966), and Te- 647-680. rashita (1970) reported that the mycangia of Sirex Gaut, I. P.C. 1969. Identity of the fungal symbiont of Sirex cyaneus Fab., S. juvencus L., S. nitobei Matsumura, S. noctilio. Aust. J. Biol. Sci. 22: 905-914. Gaut, I. P. C. 1970. Studies of siricids and their fungal sym- noctilio Fab., Urocerus albicornis Fab. and U. gigas L. bionts. PhD thesis, Univ. Adelaide, Australia. were filled with arthrospores. However, we found the Ginns, J. and Lefebvre, M. N. L. 1993. Lignicolous corticioid fungus in the mycangia of U. japonicus to be present as fungi (Basidiomycota) of North America, Systematics, distri- hyphal fragments, in which cells were not separate but bution, and ecology. Mycologia Mem., No. 19. Minnesota. joined by clamp connections. Hayashi, Y. 1974. Studies on the genus Peniophora Cke. and Sano et al. (1995) examined the fungus from the its allied genera in Japan. Bull Gov. For. Exp. Sta. 260: mycangia of U. japonicus in Mie Prefecture in the Kinki 1-98, District of Japan and reported it to be as A. chailletii. Kanamitsu, K. 1978. Woodwasps and their hymenopterous The finding of different fungal species associated with parasitoids in Japanese conifers. Kontyu 46: 498-508. (In the same species of horntail conflicts with the report of Japanese.) Kitajima, K. 1938. Forest pathology (Jubyogaku-oyobi- Gaut (1970), who showed that the same horntail species mokuzaifukyuron), pp. 442-443. Yokendo, Tokyo. (In were always associated with the same fungal species, ir- Japanese.) respective of geographical distribution. Therefore, fur- Nishiguchi, H., Shibata, E., and Yamanaka, K. 1981. Discolora- ther studies are required to clarify the species of fungus tion of sugi (Cryptomeria japonica) wood caused by the at- associated with the Japanese horntail in different dis- tack of the horntails. Trans. 32th Meeting Kansai Br. Jpn. tricts of Japan. For. Soc., pp. 257-260. (In Japanese.) Inoculation of isolates from a mycangium and a Okuda, K. 1985. Discoloration of hinoki (Chamaecyparis obtu- basidiocarp resulted in wood discoloration in all inoculat- sa) wood caused by the attack of the Japanese horntail ed trees of Ch. obtusa and Cr. japonica. The discolora- (Urocerusjaponicus). Trans. 96th Meeting Jpn. For. Soc., tion reached 28.5 cm longitudinally, 0.8 cm tangentially, pp. 487-488. (In Japanese.) and 2.4cm radially. From this result, it was able to Okuda, M. 1989. Japanese horntail. Ringyo-to-yakuzai 108: 1-8. (In Japanese.) show the damage of wood discoloration again. No Sano, A. 1992. Japanese hornteil. Ringyo-to-yakuzai 122: difference in wood discoloration was found between the 1-8. (In Japanese.) two isolates inoculated on trees of the same species. Sano, A., Mihara, Y. and Ito, S. 1995. The fungus isolated However, the extent of discoloration was larger in Ch. from the mycangia of Urocerus japonicus and U. antenna- obtusa than in Cr. japonica, especially in the longitudinal tus. Trans. 43th Meeting Chubu Br. Jpn. For. Soc, direction. The results were in close accord with those pp. 125-126. (In Japanese.) reported by Sano et al. (1995) and Suto (1994). Stalpers, J. A. 1978. Identification of wood-inhabiting Aphyllo- phorales in pure culture. Stud. Mycol. (CBS) 16: 1-248. Acknowledgements---The authors are grateful to Mr. H. Miya- Stillwell, M.A. 1966. Woodwasps (Siricidae) in conifers and ta, Kochi Prefectural Forest Experiment Station, Mr. M. Ookubo, the associated fungus, Stereum chailletii, in Eastern Cana- Kagawa Forest Center, and Mr. K. Inoue, Ehime Prefectural da. For. Sci. 12: 121-128. Forest Experiment Station for help in collecting the felled logs of Suto, Y. 1994. Wood stain caused by Amylostereum sp. in Chamaecyparis obtusa and Cryptomeriajaponica. Cryptomeria japonica and Chamaecyparis obtusa, Influence of life and death of the trees on the stain occurrence end fungal survival. Trans. Kansai Br. Jpn. For. Soc. 3: Literature cited 163-164. (In Japanese.) Takeuchi, K. 1962. Insecta Japonica: Siricidae, pp. 1-12. Boiden, J. 1958. H~t~robasidiomyc~tes saprophytes et Homo- Hokuryukan, Tokyo. (In Japanese.) basidiomyc~tes r6supin6s: V. Essai sur le genre Strereum Terashita, T. 1970. A Basidiomycetes symbiotic to a siricid in Pers. ex S. F. Gray. Rev. Mycol. 24: 197-225. Japan. J. Jpn. For. Soc. 52: 313-316. (In Japanese.)